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Hybridization chain reaction (HCR) for amplifying nanopore signals
Journal Pre-proof Hybridization chain reaction (HCR) for amplifying nanopore signals Hong Sun, Fujun Yao, Zhuoqun Su, Xiao-Feng Kang PII:
S0956-5663(19)30985-6
DOI:
https://doi.org/10.1016/j.bios.2019.111906
Reference:
BIOS 111906
To appear in:
Biosensors and Bioelectronics
Received Date: 15 October 2019 Revised Date:
6 November 2019
Accepted Date: 18 November 2019
Please cite this article as: Sun, H., Yao, F., Su, Z., Kang, X.-F., Hybridization chain reaction (HCR) for amplifying nanopore signals, Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/ j.bios.2019.111906. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Hybridization chain reaction (HCR) for amplifying nanopore signals
Hong Sun†, FujunYao†, Zhuoqun Su and Xiao-Feng Kang*
Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an 710069, P. R. China
† These authors contributed equally to this work. * Corresponding author. Fax: +86-029-88302604. Phone: +86-029-88302604. E-mail: [email protected]
Abstract Circulating tumor DNA (ctDNA) in the blood is an important biomarker for noninvasive diagnosis, assessment, prediction and treatment of cancer. However, sensing performance of solid nanopore is limited by the fast kinetics of small DNA targets and unmatched dimensions. Here, we combines hybridization chain reaction (HCR) with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer. The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore, but also significantly elevates both selectivity and signal-to-noise ratio, which allows to detect ctDNA at a detection limit of 2.8 fM (S/N=3) and the single-base resolution. Furthermore, the proposed method can apply in detection of ctDNA of KRAS G12DM in serum sample.
Keywords: Circulating tumor DNA; Solid nanopore; Hybridization chain reaction; Singlemolecule analysis; Amplification detection; Biomarker.
1. Introduction Almost half of people who get cancer are diagnosed late, which makes therapeutic intervention less likely to succeed and reduces their chances of survival. Unfortunately, there are not broadly available biomarkers that have been clinically approved and can widely be employed to early diagnose and manage patients with cancer. Circulating tumor DNA (ctDNA) with genetic mutations or genetic changes is a fraction of cell-free DNA (cfDNA) derived from tumors (Chimonidou et al. 2011; Li et al. 2019). ctDNA is present as single-stranded or doublestranded DNA in peripheral blood. Recently, whole-genome sequencing (WGS) analysis of cfDNA has indicated that the strategy to detect tumor-specific alterations has the potential to become a noninvasive liquid biopsy for early diagnosis (Chi 2016; Cristiano et al. 2019; Ignatiadis et al. 2015). Moreover, Short the half-life of ctDNA (less than 2 h), in contrast to that of most protein biomarkers (several weeks), could make a much clearer diagnosis through realtime tumor dynamic monitoring (Zhou et al. 2016). Although ctDNA is particularly informative as a biomarker, specific routine analysis of ctDNA in the circulation has many challenges. Owing to extremely low number of abnormal alteration, identifying such tumor-specific abnormalities among bulk cfDNA is one of challenges (Jiang et al. 2015; Leary et al. 2012). Recent works have demonstrated that the selection and analysis of small DNA fragmentation can increase enrichment of ctDNA in patients with cancers, which is called as fragmentation method (Snyder et al. 2016; Ulz et al. 2016; Underhill et al. 2016). A typical blood sample contains about 2,000 genome equivalents of cfDNA per milliliter of plasma (Phallen et al. 2017). To extract the information of the ctDNA distribution, whole-genome sequencing of such a large number of samples is another huge challenge. Therefore, a fast, simple, sensitive and reliable PCR-free technology for detecting small specific DNA fragmentation is urgently required.
Nanopore sensing is a powerful single-molecule technology that replies on the changes in nanopore current produced by analyte-translocating events. Mechanically stable solid-state nanopores are ideal for clinical and commercial applications. In view of many merits such as simplicity, label-free, solution-based detection and easy operation, solid-state nanopores have been developed for exploring protein-DNA interactions (Jeong et al. 2018), protein-protein interactions (Tiwari et al. 2014), and DNA nanostructure (Zhu et al. 2018) by the translocation signals. But, in sensing small molucules like short DNA fragmentation, it is unsuccessful because of the fast transport kinetics and unmatched dimensions. Some strategies have been proposed to improve the inherent limitations including lipid-coating (Yusko et al. 2011), DNA modification (Carlsen et al. 2014), carriers of DNA (Bell and Keyser 2015) and gold nanoparticle (Lin et al. 2017). These approaches are cumbersome, require careful optimization, yet frequently are ineffective. In this work, we introduce hybridization chain reaction (HCR) (Dirks and Pierce 2004; Lin et al. 2018) into nanopore sensing system for the first time to detect ctDNA. As a dynamic DNA self-assemble nanotechnology (Dirks and Pierce 2004), HCR has been applied to construct efficient enzyme-free amplification platform in biosensing of DNA (Yue et al. 2017) and MicroRNA (Bi et al. 2016; Yue et al. 2019). Small target ssDNA fragment triggers HCR to amplify the short ssDNA strand into long nicked double helics concatemer, permitting solid nanopore to implement single-molecule measurements. Moreover, to demonstrate the feasibility of the strategy in real sample analysis, we choose a single stranded fragment KRAS G12DM as the model ctDNA. It has been reported that the expression level of KRAS G12DM is closely associated with colon carcinoma disease and could be employed as a diagnostic biomarker of cancer progression (Zhou et al. 2016; Diehl et al. 2006). The amplification achieved by coupling
HCR to solid nanopore offers an enzyme-free single-molecule technology for the rapid detection of small specific DNA sequences, greatly extending the application fields of solid nanopore. 2. Experimental section 2.1. Materials and regents The poly (ethylene terephthalate) (PET) membranes (12 µm in thick) were purchased from GSI in Darmstadt, Germany. Lyophilized oligonucleotides designed in this study were synthesized and purified by Sangon Biotech Company, Ltd. (Shanghai, China), and their sequences are listed in Table S1. Human serum (H4522) was provided by Sigma-Aldrich. All other reagents were of analytical grade and used as received. 2.2. Fabrication and characterization of nanochannel The conical PET nanochannel was prepared by the well-developed ion track etching technique (Wharton et al. 2007). The prepared conical nanochannel has two openings. The diameter of large opening (terms as the base) was approximately 410 nm, which was measured by scanning electron microscopy (SEM). The diameter of small opening (terms as the tip) was calculated to be ~6 nm through electrochemical measurements (Wharton et al. 2007) (details see the Supporting Information). 2.3. Nanopore translocation experiments The conical polymeric nanochannel separated the cells into two isolated containers. The tip side of nanochannel acted as the cis reservoir (connected to “ground”), while the base side acted as the trans reservoir. Both reservoirs were filled with 1 M KCl (10 mM Tris-HCl, buffered at pH 7.0). For the ctDNA detection, a series of concentrations of target ctDNA were mixed with auxiliary probes solution and were incubated for 2 h at 37 °C (details operation see the
Supporting Information). Subsequently, the reaction solution was added to the base side reservoir, and a voltage of - 1.0 V (base side) was applied to drive the DNAs translocation through the nanochannel. A patch clamp amplifier (Axopatch 200B, Molecular Devices Inc.) was employed to measure and synchronously record the current trace across the conical nanochannel. The resulting current data were filtered by a low-pass Bessel filter of 5 kHz, sampled at 20 kHz by a computer equipped with a Digidata 1550 converter (Molecular Devices) and acquired with Clampex 10.5 software (Molecular Devices). The root mean square (RMS) value of the measurements in this experiment is less than 3 pA. 2.4. Data analysis The event amplitude and duration were analyzed by using Clampfit 10.5 (Molecular Devices) and origin 9.0 software (Microcal, Northampton, MA). The values of average pulse amplitude (∆I) were obtained from ∆I histograms by fitting the distributions to Gaussian functions. Mean dwell time values (τoff) and mean interevent interval values (τon) were obtained from the dwell histograms and interevent histograms which were fitted to single exponential functions, respectively. The event frequency (1/τon) was obtained from the 1/τon histograms by fitting the distributions to single exponential functions. 2.5. Gel Electrophoresis 1% agarose gel was prepared using 1 × TAE buffer (40 mM Tris-AcOH, 2.0 mM Na2EDTA, pH 8.5). The GoldView was used as an oligonucleotide dye. The gel was run at 42 V for 150 min in 1× TAE buffer with loading of 10 µL of the sample into each lane at room temperature. Afterward, it was photographed using the gel image analysis system (Tanon 2500R, Tianneng Ltd, Shanghai, China).
3. Results and discussion 3.1. Sensor design Fig. 1A is the schematic of HCR-nanopore for DNA detection. In this HCR system, H1 and H2 are two auxiliary hairpin probes, which can coexist and do not bind each other in solution. But when a single-stranded target DNA initiator is added to the stable mixture, it opens a hairpin of one species, exposing a new single-stranded region that opens a hairpin of the other species. In such a way, two hairpin probes are alternatively unlocked to polymerize into a long-nicked duplex DNA concatemer (Bi et al. 2017). In the nanopore detector, a conical nanochannel is employed as sensing element, which was fabricated from PET foil using an asymmetry ion track etching technique (the details see experimental section). The diameter of large opening (base) is approximately 410 nm, as determined by the SEM imaging (Fig. 1C), while the small opening (tip) is 6 nm by electrochemical measurement (Wharton et al. 2007) (Fig. 1D). For the short target DNA, H1 and H2, the solid nanopore cannot produce electronic signal. But by the combination of HRC-nanopore, the long DNA concatemer, as the substitution of the short target DNA, results in high-resolution pulse signal. The strategy significantly elevates the signal-tonoise ratio, permitting the solid nanopore to detect small specific DNA sequences.
Figure 1 should be inserted in here
3.2. DNA nanopore signatures The nanopore current was recorded at an applied voltage of - 1.0 V in 10 mM Tris-HCl, buffer (pH 7.0) containing 1.0 M KCl. The typical traces of different DNAs are shown in Fig. 1A. We
observe that the target DNA does not generate any blocking current (Right-top trace in Fig. 1A), suggesting that the pore is too inert to sense such short single-strand DNA. For the mixture of two auxiliary hairpin probes (H1 and H2), only very few and short events appear during the entire recording (Right-middle trace in Fig. 1A). However, as expected, after adding the target DNA to the mixture of H1 and H2, the long characteristic signals are produced (Right-bottom trace in Fig. 1A). We attribute the distinctive events to the formation and subsequent nanochannel translocation of long dsDNA concatemer. We further investigate the translocation of dsDNA concatemer in the nanochannel at different voltages, ranging from - 0.4 V to - 1.0 V. The results show that the event frequency increases exponentially as the applied voltage increases (Fig. 1B). Therefore, - 1.0 V was chosen as the optimized applied potential and used in the following experiments. The observed pulse signal is in agreement with previous studies regarding the translocation of long dsDNA carrier through a glass nanopore (Chen et al. 2017). The formation of the long DNA polymers was further confirmed by gel electrophoresis (Fig. S1). By examining the detail of DNA concatemer signals, we found that three distinct current patterns (Fig. 2A, bottom) were produced during threading the DNA through the pore: current decrease (type 1), current increase (type 2) and biphasic pulse with the current first decreasing and then increasing (type 3). The current signals of nanopore with different shapes are often explained by two main factors: (i) volume exclusion effect and (ii) the surface charge effect (Chen et al. 2017). In the present work, we deduce that three current patterns are ascribed to the combined effects that result in three types of translocation fashion (Fig. 2A, top). The signals of type 1, with a small fraction of the pulses (~16 %), are probably generated from the DNA polymers translocation through the pore in a fully folded structure (Fig.2A, left) in which the volume exclusion effect plays a dominant role. For the signals of type 2 (~24 % of the total
events), the increase in ion conductance can be associated with the linear threading of the DNA concatemers from head-to-tail fashion without folds (Fig.2A, middle). The excess ions that the DNA polymer brings into pore during translocation are responsible for the current increase. The results are consistent with previous studies which attributed to an increase in nanopore current resulting from the charge of DNA and its mobile counterions (Smeets et al. 2006). As for the biphasic pulse of type 3, which happens for a large majority of events (~60 %), we speculate that the signals are corresponding to the translocation of DNA concatemers in a partially folded manner, which is analogue to a hairpin DNA structure with an overhang (Fig. 2A, right). The folded end first enters the nanochannel and thus the dominant volume exclusion effect leads to a current decrease. As the translocation process goes on, the rest unfold part start entering the sensing zone of the nanochannel (usually the first ~1 µm zone from the tip) (Sexton et al. 2010). At this moment, the effect of ion concentration enhancement exceeds the volume exclusion effect, leading to a current increase. The findings are in excellent agreement with prior studies of Chen and co-workers (Chen et al. 2017). They have demonstrated that backward translocation of dsDNA through the pore could produce biphasic pulses in 1 M KCl solution at negative voltage. Our interpretations can also be supported by a previous work that proposed that long dsDNA translocate through nanopore is not only from head to tail, but also in a fold fashion (Merchant et al. 2010; Zhu et al. 2019). The folding DNA translocation events are further proved by analyzing current amplitude. Fig. 2B shows the histogram of the current changes from 2308 of translocation events, in which four peaks are vividly presented at - 37.52 pA, - 17.50 pA, 17.01 pA and 38.73 pA, as indicated by the Gaussian fits. These peaks clearly indicate the folding phenomenon of the DNA concatemers during passing through the nanopore (Bell et al. 2012). In addition to the event amplitude, we
also studied the dwell times in the pore. As depicted in Fig. 2C, the average values of dwell time for the current levels I, II, III and IV are 0.65 ± 0.05 ms, 0.34 ± 0.01 ms, 0.38 ± 0.01 ms, and 0.76 ± 0.09 ms, respectively. Obviously, the dwell time is highly dependent on the DNA length in the pore. The longest unfolding dsDNA concatemer has the largest dwell time.
Figure 2 should be inserted in here
3.3. Optimization of HCR The nanopore ctDNA biosensor proposed here is constructed based on forming the long dsDNA concatemer, and hence increasing the event frequency of the DNA concatemer in the nanopore can greatly improve the detection limit of ctDNA and the resolution of the nanopore sensor. It has been well documented that the molecular weight of HCR product is closely related with the amount of the initiator (Lu et al. 2017). In order to optimize the reaction conditions, we explored the molar concentration ratio of the initiator (I0) and the hairpin probe H1, [I0]/[H1], which can directly control the length distribution of DNA concatemers. Fig. 3A gives the representative current traces under different [I0]/[H1] ratios, ranging from 1:10 to 1:2000. At [I0]/[H1]= 1:10, the major events are the signals of type 2. With dropping the ratio, the percent of type 2 first decreases and then keeps unchanged, while the signals of both type 1 and type 3 became main events. The results are not unreasonable considering the length distribution of DNA concatemers at various [I0]/[H1]. According to previous studies, the molecular weight of HCR products is inversely proportional to the amount of initiator (Lu et al. 2017). Therefore, a decrease in the ratio of [I0]/[H1] would lead to an increase in the length of DNA concatemers. As discussed in the previous section, the long-range DNA polymers are readily to translocate
through the nanopore in a folded fashion under a high applied voltage, thus producing current decrease or biphasic pulse signals. These results were further confirmed by the gel electrophoresis (Fig. 3B). The control experimental result demonstrates that only one band is observed at very low position in the absence of target DNA, confirming that the two hairpin probes stably coexist in the solution (lane 7). However, a series of new bright stripes are generated in the presence of various ratios of [I0]/[H1], indicating that the HCR reaction have taken place and the two auxiliary probes had been assembled to long-nicked DNA concatemers (lane 1-6). Interestingly, the position of these stripes, corresponding to the molecular weight of assembly products, changes with the concentration of H1 and H2 when the concentration of target DNA was constant. At the high ratios of 1:10 and 1:20, the molecular weight of longnicked DNA polymers is relatively small since plentiful target DNA compete the hairpin DNAs and restricted the assembly degree of HCR (lane1-2). When the ratio decreases to 1:100 or lower (lane 3-4), the stripes at lower position became dim, while new bright stripes are observed at higher position. The findings suggest that the DNA polymerization reaches the maximum degree and the molecular weight of DNA concatemers were relatively large. However, the molecular weight of self- assembled DNA concatemers are slightly decreased when the concentration of H1 and H2 increases from 10 µM to 20 µM (lane 5-6), it could be explained by a reported “inhibitor ultrasensitivity” mechanism (Li et al. 2012). It is worth to mention that the molecular weight of the HCR products were an approximate range, not an exact numerical value (Liu et al. 2013). Therefore, multiple pulse signals could be produced during the translocation of the products. The dependence of the event frequency on the ratio of [I0]/[H1] is shown in Fig. 3C. Interestingly, the characteristic signals frequency exhibits a non-monotonic dependence with decreasing the ratio of [I0]/[H1]. As the [I0]/[H1] decreases from 1:10 to1:100, the event
frequency increases from 48 ± 2.3 to 58 ± 2.7 min-1. Then a gradual decrease is observed in the range of 1:100 to 1:2000. In addition, the effect of the ratio of [I0]/[H1] on current pulse amplitude was also examined. As shown in Fig. 3D, the pulse amplitude first shows a slight increase and then remains almost constant as an increase in the ratio. These results could be explained by the fact that the molecular mass of the DNA concatemers products reaches a summit at a specific ratio of [I0]/[H1], 1:100. This interpretation can be also supported by the gel electrophoresis experiment as shown in Fig. 3B. In order to achieve the detection of target DNA with high sensitivity and resolution, the ratio of 1:100 is chosen as the optimized condition and used in the remaining experiments.
Figure 3 should be inserted in here
3.4. Detection sensitivity and specificity Fig. 4A is representative current traces at various target DNA concentrations. The frequency of characteristic signals increases with an increase in the concentration of target DNA in a wide range of 20 fM to 5 pM (Fig. 4B). Linear regression analysis exhibits good linearity in the range of 50 fM-1.0 pM, with a correlation coefficient of 0.994 (Fig. 4B, inset). The limit of detection is 5.2 fM (S/N = 3). To evaluate the selectivity, a series of comparative studies using single-base mismatch (MT1), two-base mismatch (MT2) and non-complementary (NC) sequences were investigated as control experiments. As illustrated in Fig. 4C, only fully matched DNA (TD) initiates the HCR and nanopore responses, while the other mismatched DNAs are difficult to trigger the reaction and to generate significant current pulse signals. The high selectivity with
single-base resolution should originate from HCR that requires full complementarity between the target DNA and hairpin auxiliary probes (Liu et al. 2013). In addition, the sequence specificity of this DNA assay was further validated by gel electrophoresis (Fig. 4D).
Figure 4 should be inserted in here
3.5. ctDNA detection It is well known that the release of KRAS G12DM, a single-stranded ctDNA into the blood is related to the apoptosis and necrosis of cancer cells in tumor tissues (Fig. 5A), and its level in blood can be used to evaluate the progression of colorectal cancer (Schwarzenbach et al. 2011). Fig. 5B shows the process of detecting KRAS G12DM ctDNA. According to KRAS G12DM sequence (see Table S1), two new hairpin auxiliary probes H3 and H4 (the sequences see Table S1) were designed, which can form long dsDNA polymers through HCR in the presence of KRAS G12DM target DNA. As observed in Fig. 5C, the characteristic signals are generated in the range of 20 fM to 2 pM. Moreover, the full matched KRAS G12DM target can be well discriminated from that of one-base, two-base and three-base mismatched DNA sequences (see Table S1 and Fig. S2 in Supporting Information). The limit of detection (LOD) was 2.8 fM, with a linear range of 20 fM-1 pM (Fig. 5D, inset). The linear regression equation can be expressed as freq=82.31[ctDNA] (pM) + 1.84 (R = 0.995). In addition, despite multicomponent (proteins and ctDNA) in serum sample, no signal was observed in the commercial serum sample (Figure. S3). This result could be attributed to a fast translocation of each individual component and thus the event signals can hardly be captured.
Figure 5 should be inserted in here
3.6. Serum Sample Assay We explored the application for detecting ctDNA in real serum. 10-fold the diluted commercial healthy human serum and the serums with added target KRAS G12DM ctDNA (100 fM, 500 fM and 1 pM) were tested by our method (Fig. S3). The detected concentrations are 90.9 ± 3.12 fM, 475.0 ± 16.76 fM and 931.1 ± 38.83 fM, respectively. The corresponding recoveries are 90.92 ± 3.12%, 95.0 ± 3.35% and 93.1 ± 3.88%, exhibiting adequate and acceptable accuracy and anti-interference ability for ctDNA detection (Table S2). 4. Conclusions Overall, we have successfully constructed HCR-nanopore sensing system and applied in the sensitive detection of ctDNA. The HCR can be harnessed in solid nanopore sensing, overcoming the limitation that has precluded the short DNA or small target. The HCR amplification is a key for obtaining high sensitivity and high selectivity. The amplitude and frequency of nanopore signal can be modulated in accordance with the target by the probe concentration. This simplest single-molecule analytical technique is very suitable for monitoring ctDNA level in circulating system that needs readout the result quickly because of short the half-life. Our approach could be further improved through single-stranded DNA libraries (Burnham et al. 2016). Combined with PCR-free libraries, the HCR-nanopore could reveal the molecular features and the origin characteristics of ctDNA that are distinct at different positions such as nucleosome positions, the start sites and the end positions of transcription (Jiang et al. 2018). Additionally, the solution-
based technology required only a small amount of blood sample (<10 µL), which should be broadly employed in the screening and management in clinical routine test.
CRediT authorship contribution statement Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was financially supported by the National Science Foundation of China (Grants 21375104, 21327806 and 21874107), and Natural Science Basic Research Plan in Shaanxi Province of China (Grant 2016JQ2021).
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Fig. 1. (A) Schematic illustration of the HCR-nanopore biosensor for DNA detection. Through the hairpin probes of H1 and H2, HCR amplifies short target DNA to form long-nicked dsDNA concatemer, followed by nanopore sensing. H1, H2 and the target were added to the base side of nanochannel. (B) Event frequency values of target DNA (black), H1/H2 (red) and DNA concatemers (blue) at different voltages. (C) SEM image of the base side of conical PET nanochannel. (D) I-V characteristics of the conical nanochannel measured in 10 mM Tris-HCl buffer (pH 7.0) containing 1 M KCl.
Fig. 2. (A) The schematic of DNA concatemers translocation processes (top), the expanded view of three typical translocation event signals (bottom). (B) The histogram of the current changes. (C) The histograms of the dwell time for four levels (Ⅰ, Ⅱ, Ⅲ and Ⅳ, as denoted in Fig. 2A). Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied voltage of - 1.0 V.
Fig. 3. (A) Typical current-time traces at various ratios of [I0]/[H1] (1:10 to 1:2000). (B) The image of gel electrophoresis (M: marker; lanes 1-6: the concentration of target DNA is 10 nM, the concentrations of H1 and H2 all are 100 nM, 200 nM, 1 µM, 5 µM, 10 µM, 20 µM, respectively; lane 7: 20 µM H1 + 20 µM H2). (C-D) The effect of molar concentration ratios of [I0]/[H1] on the frequency (C) and amplitude (D). Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied potential of - 1.0 V.
Fig. 4. (A) Representative current traces of target DNA at various concentrations. (B) The plots of translocation event frequency versus the target DNA concentrations. Inset is the linear relationship between the event frequency and the target DNA concentration in the range of 50 fM to 1 pM. (C) Selectivity of the method for target DNA detection. The concentration of target DNA and the mismatched DNA sequences (see Table S1) are 1 pM and 10 pM, respectively. (D) Agarose electrophoresis images (M: marker; lane 1: H1 + H2; lane 2: H1 + H2 + target DNA; lane 3: H1 + H2 + MT1; lane 4: H1 + H2 + MT2; Lane 5: H1 + H2 + NC. The concentration of target sequence and auxiliary probes were 10 nM and 1 µM, respectively).
Fig. 5. (A) Schematic illustration of circulating tumor DNA fragments releasing from cells and circulating in the blood. (B) The analysis process of KRAS G12DM ctDNA. (C) The typical current traces of KRAS G12DM ctDNA with different concentrations. (D) Dose-response curve of KRAS G12DM ctDNA. Inset is the linear calibration curve. Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied potential of - 1.0 V.
Highlights
1. We have constructed HCR-nanopore sensing system and applied in the detection of ctDNA. 2. The sensing strategy exhibited well selective and the detection limit is 2.8 fM. 3. The proposed method can apply in detection of ctDNA in serum sample. 4. The method potentially offers a simple and non-invasive liquid biopsy for diagnosing cancer.
Author Contribution Statement
Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
CRediT authorship contribution statement Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0956-5663(19)30985-6
DOI:
https://doi.org/10.1016/j.bios.2019.111906
Reference:
BIOS 111906
To appear in:
Biosensors and Bioelectronics
Received Date: 15 October 2019 Revised Date:
6 November 2019
Accepted Date: 18 November 2019
Please cite this article as: Sun, H., Yao, F., Su, Z., Kang, X.-F., Hybridization chain reaction (HCR) for amplifying nanopore signals, Biosensors and Bioelectronics (2019), doi: https://doi.org/10.1016/ j.bios.2019.111906. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Hybridization chain reaction (HCR) for amplifying nanopore signals
Hong Sun†, FujunYao†, Zhuoqun Su and Xiao-Feng Kang*
Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an 710069, P. R. China
† These authors contributed equally to this work. * Corresponding author. Fax: +86-029-88302604. Phone: +86-029-88302604. E-mail: [email protected]
Abstract Circulating tumor DNA (ctDNA) in the blood is an important biomarker for noninvasive diagnosis, assessment, prediction and treatment of cancer. However, sensing performance of solid nanopore is limited by the fast kinetics of small DNA targets and unmatched dimensions. Here, we combines hybridization chain reaction (HCR) with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer. The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore, but also significantly elevates both selectivity and signal-to-noise ratio, which allows to detect ctDNA at a detection limit of 2.8 fM (S/N=3) and the single-base resolution. Furthermore, the proposed method can apply in detection of ctDNA of KRAS G12DM in serum sample.
Keywords: Circulating tumor DNA; Solid nanopore; Hybridization chain reaction; Singlemolecule analysis; Amplification detection; Biomarker.
1. Introduction Almost half of people who get cancer are diagnosed late, which makes therapeutic intervention less likely to succeed and reduces their chances of survival. Unfortunately, there are not broadly available biomarkers that have been clinically approved and can widely be employed to early diagnose and manage patients with cancer. Circulating tumor DNA (ctDNA) with genetic mutations or genetic changes is a fraction of cell-free DNA (cfDNA) derived from tumors (Chimonidou et al. 2011; Li et al. 2019). ctDNA is present as single-stranded or doublestranded DNA in peripheral blood. Recently, whole-genome sequencing (WGS) analysis of cfDNA has indicated that the strategy to detect tumor-specific alterations has the potential to become a noninvasive liquid biopsy for early diagnosis (Chi 2016; Cristiano et al. 2019; Ignatiadis et al. 2015). Moreover, Short the half-life of ctDNA (less than 2 h), in contrast to that of most protein biomarkers (several weeks), could make a much clearer diagnosis through realtime tumor dynamic monitoring (Zhou et al. 2016). Although ctDNA is particularly informative as a biomarker, specific routine analysis of ctDNA in the circulation has many challenges. Owing to extremely low number of abnormal alteration, identifying such tumor-specific abnormalities among bulk cfDNA is one of challenges (Jiang et al. 2015; Leary et al. 2012). Recent works have demonstrated that the selection and analysis of small DNA fragmentation can increase enrichment of ctDNA in patients with cancers, which is called as fragmentation method (Snyder et al. 2016; Ulz et al. 2016; Underhill et al. 2016). A typical blood sample contains about 2,000 genome equivalents of cfDNA per milliliter of plasma (Phallen et al. 2017). To extract the information of the ctDNA distribution, whole-genome sequencing of such a large number of samples is another huge challenge. Therefore, a fast, simple, sensitive and reliable PCR-free technology for detecting small specific DNA fragmentation is urgently required.
Nanopore sensing is a powerful single-molecule technology that replies on the changes in nanopore current produced by analyte-translocating events. Mechanically stable solid-state nanopores are ideal for clinical and commercial applications. In view of many merits such as simplicity, label-free, solution-based detection and easy operation, solid-state nanopores have been developed for exploring protein-DNA interactions (Jeong et al. 2018), protein-protein interactions (Tiwari et al. 2014), and DNA nanostructure (Zhu et al. 2018) by the translocation signals. But, in sensing small molucules like short DNA fragmentation, it is unsuccessful because of the fast transport kinetics and unmatched dimensions. Some strategies have been proposed to improve the inherent limitations including lipid-coating (Yusko et al. 2011), DNA modification (Carlsen et al. 2014), carriers of DNA (Bell and Keyser 2015) and gold nanoparticle (Lin et al. 2017). These approaches are cumbersome, require careful optimization, yet frequently are ineffective. In this work, we introduce hybridization chain reaction (HCR) (Dirks and Pierce 2004; Lin et al. 2018) into nanopore sensing system for the first time to detect ctDNA. As a dynamic DNA self-assemble nanotechnology (Dirks and Pierce 2004), HCR has been applied to construct efficient enzyme-free amplification platform in biosensing of DNA (Yue et al. 2017) and MicroRNA (Bi et al. 2016; Yue et al. 2019). Small target ssDNA fragment triggers HCR to amplify the short ssDNA strand into long nicked double helics concatemer, permitting solid nanopore to implement single-molecule measurements. Moreover, to demonstrate the feasibility of the strategy in real sample analysis, we choose a single stranded fragment KRAS G12DM as the model ctDNA. It has been reported that the expression level of KRAS G12DM is closely associated with colon carcinoma disease and could be employed as a diagnostic biomarker of cancer progression (Zhou et al. 2016; Diehl et al. 2006). The amplification achieved by coupling
HCR to solid nanopore offers an enzyme-free single-molecule technology for the rapid detection of small specific DNA sequences, greatly extending the application fields of solid nanopore. 2. Experimental section 2.1. Materials and regents The poly (ethylene terephthalate) (PET) membranes (12 µm in thick) were purchased from GSI in Darmstadt, Germany. Lyophilized oligonucleotides designed in this study were synthesized and purified by Sangon Biotech Company, Ltd. (Shanghai, China), and their sequences are listed in Table S1. Human serum (H4522) was provided by Sigma-Aldrich. All other reagents were of analytical grade and used as received. 2.2. Fabrication and characterization of nanochannel The conical PET nanochannel was prepared by the well-developed ion track etching technique (Wharton et al. 2007). The prepared conical nanochannel has two openings. The diameter of large opening (terms as the base) was approximately 410 nm, which was measured by scanning electron microscopy (SEM). The diameter of small opening (terms as the tip) was calculated to be ~6 nm through electrochemical measurements (Wharton et al. 2007) (details see the Supporting Information). 2.3. Nanopore translocation experiments The conical polymeric nanochannel separated the cells into two isolated containers. The tip side of nanochannel acted as the cis reservoir (connected to “ground”), while the base side acted as the trans reservoir. Both reservoirs were filled with 1 M KCl (10 mM Tris-HCl, buffered at pH 7.0). For the ctDNA detection, a series of concentrations of target ctDNA were mixed with auxiliary probes solution and were incubated for 2 h at 37 °C (details operation see the
Supporting Information). Subsequently, the reaction solution was added to the base side reservoir, and a voltage of - 1.0 V (base side) was applied to drive the DNAs translocation through the nanochannel. A patch clamp amplifier (Axopatch 200B, Molecular Devices Inc.) was employed to measure and synchronously record the current trace across the conical nanochannel. The resulting current data were filtered by a low-pass Bessel filter of 5 kHz, sampled at 20 kHz by a computer equipped with a Digidata 1550 converter (Molecular Devices) and acquired with Clampex 10.5 software (Molecular Devices). The root mean square (RMS) value of the measurements in this experiment is less than 3 pA. 2.4. Data analysis The event amplitude and duration were analyzed by using Clampfit 10.5 (Molecular Devices) and origin 9.0 software (Microcal, Northampton, MA). The values of average pulse amplitude (∆I) were obtained from ∆I histograms by fitting the distributions to Gaussian functions. Mean dwell time values (τoff) and mean interevent interval values (τon) were obtained from the dwell histograms and interevent histograms which were fitted to single exponential functions, respectively. The event frequency (1/τon) was obtained from the 1/τon histograms by fitting the distributions to single exponential functions. 2.5. Gel Electrophoresis 1% agarose gel was prepared using 1 × TAE buffer (40 mM Tris-AcOH, 2.0 mM Na2EDTA, pH 8.5). The GoldView was used as an oligonucleotide dye. The gel was run at 42 V for 150 min in 1× TAE buffer with loading of 10 µL of the sample into each lane at room temperature. Afterward, it was photographed using the gel image analysis system (Tanon 2500R, Tianneng Ltd, Shanghai, China).
3. Results and discussion 3.1. Sensor design Fig. 1A is the schematic of HCR-nanopore for DNA detection. In this HCR system, H1 and H2 are two auxiliary hairpin probes, which can coexist and do not bind each other in solution. But when a single-stranded target DNA initiator is added to the stable mixture, it opens a hairpin of one species, exposing a new single-stranded region that opens a hairpin of the other species. In such a way, two hairpin probes are alternatively unlocked to polymerize into a long-nicked duplex DNA concatemer (Bi et al. 2017). In the nanopore detector, a conical nanochannel is employed as sensing element, which was fabricated from PET foil using an asymmetry ion track etching technique (the details see experimental section). The diameter of large opening (base) is approximately 410 nm, as determined by the SEM imaging (Fig. 1C), while the small opening (tip) is 6 nm by electrochemical measurement (Wharton et al. 2007) (Fig. 1D). For the short target DNA, H1 and H2, the solid nanopore cannot produce electronic signal. But by the combination of HRC-nanopore, the long DNA concatemer, as the substitution of the short target DNA, results in high-resolution pulse signal. The strategy significantly elevates the signal-tonoise ratio, permitting the solid nanopore to detect small specific DNA sequences.
Figure 1 should be inserted in here
3.2. DNA nanopore signatures The nanopore current was recorded at an applied voltage of - 1.0 V in 10 mM Tris-HCl, buffer (pH 7.0) containing 1.0 M KCl. The typical traces of different DNAs are shown in Fig. 1A. We
observe that the target DNA does not generate any blocking current (Right-top trace in Fig. 1A), suggesting that the pore is too inert to sense such short single-strand DNA. For the mixture of two auxiliary hairpin probes (H1 and H2), only very few and short events appear during the entire recording (Right-middle trace in Fig. 1A). However, as expected, after adding the target DNA to the mixture of H1 and H2, the long characteristic signals are produced (Right-bottom trace in Fig. 1A). We attribute the distinctive events to the formation and subsequent nanochannel translocation of long dsDNA concatemer. We further investigate the translocation of dsDNA concatemer in the nanochannel at different voltages, ranging from - 0.4 V to - 1.0 V. The results show that the event frequency increases exponentially as the applied voltage increases (Fig. 1B). Therefore, - 1.0 V was chosen as the optimized applied potential and used in the following experiments. The observed pulse signal is in agreement with previous studies regarding the translocation of long dsDNA carrier through a glass nanopore (Chen et al. 2017). The formation of the long DNA polymers was further confirmed by gel electrophoresis (Fig. S1). By examining the detail of DNA concatemer signals, we found that three distinct current patterns (Fig. 2A, bottom) were produced during threading the DNA through the pore: current decrease (type 1), current increase (type 2) and biphasic pulse with the current first decreasing and then increasing (type 3). The current signals of nanopore with different shapes are often explained by two main factors: (i) volume exclusion effect and (ii) the surface charge effect (Chen et al. 2017). In the present work, we deduce that three current patterns are ascribed to the combined effects that result in three types of translocation fashion (Fig. 2A, top). The signals of type 1, with a small fraction of the pulses (~16 %), are probably generated from the DNA polymers translocation through the pore in a fully folded structure (Fig.2A, left) in which the volume exclusion effect plays a dominant role. For the signals of type 2 (~24 % of the total
events), the increase in ion conductance can be associated with the linear threading of the DNA concatemers from head-to-tail fashion without folds (Fig.2A, middle). The excess ions that the DNA polymer brings into pore during translocation are responsible for the current increase. The results are consistent with previous studies which attributed to an increase in nanopore current resulting from the charge of DNA and its mobile counterions (Smeets et al. 2006). As for the biphasic pulse of type 3, which happens for a large majority of events (~60 %), we speculate that the signals are corresponding to the translocation of DNA concatemers in a partially folded manner, which is analogue to a hairpin DNA structure with an overhang (Fig. 2A, right). The folded end first enters the nanochannel and thus the dominant volume exclusion effect leads to a current decrease. As the translocation process goes on, the rest unfold part start entering the sensing zone of the nanochannel (usually the first ~1 µm zone from the tip) (Sexton et al. 2010). At this moment, the effect of ion concentration enhancement exceeds the volume exclusion effect, leading to a current increase. The findings are in excellent agreement with prior studies of Chen and co-workers (Chen et al. 2017). They have demonstrated that backward translocation of dsDNA through the pore could produce biphasic pulses in 1 M KCl solution at negative voltage. Our interpretations can also be supported by a previous work that proposed that long dsDNA translocate through nanopore is not only from head to tail, but also in a fold fashion (Merchant et al. 2010; Zhu et al. 2019). The folding DNA translocation events are further proved by analyzing current amplitude. Fig. 2B shows the histogram of the current changes from 2308 of translocation events, in which four peaks are vividly presented at - 37.52 pA, - 17.50 pA, 17.01 pA and 38.73 pA, as indicated by the Gaussian fits. These peaks clearly indicate the folding phenomenon of the DNA concatemers during passing through the nanopore (Bell et al. 2012). In addition to the event amplitude, we
also studied the dwell times in the pore. As depicted in Fig. 2C, the average values of dwell time for the current levels I, II, III and IV are 0.65 ± 0.05 ms, 0.34 ± 0.01 ms, 0.38 ± 0.01 ms, and 0.76 ± 0.09 ms, respectively. Obviously, the dwell time is highly dependent on the DNA length in the pore. The longest unfolding dsDNA concatemer has the largest dwell time.
Figure 2 should be inserted in here
3.3. Optimization of HCR The nanopore ctDNA biosensor proposed here is constructed based on forming the long dsDNA concatemer, and hence increasing the event frequency of the DNA concatemer in the nanopore can greatly improve the detection limit of ctDNA and the resolution of the nanopore sensor. It has been well documented that the molecular weight of HCR product is closely related with the amount of the initiator (Lu et al. 2017). In order to optimize the reaction conditions, we explored the molar concentration ratio of the initiator (I0) and the hairpin probe H1, [I0]/[H1], which can directly control the length distribution of DNA concatemers. Fig. 3A gives the representative current traces under different [I0]/[H1] ratios, ranging from 1:10 to 1:2000. At [I0]/[H1]= 1:10, the major events are the signals of type 2. With dropping the ratio, the percent of type 2 first decreases and then keeps unchanged, while the signals of both type 1 and type 3 became main events. The results are not unreasonable considering the length distribution of DNA concatemers at various [I0]/[H1]. According to previous studies, the molecular weight of HCR products is inversely proportional to the amount of initiator (Lu et al. 2017). Therefore, a decrease in the ratio of [I0]/[H1] would lead to an increase in the length of DNA concatemers. As discussed in the previous section, the long-range DNA polymers are readily to translocate
through the nanopore in a folded fashion under a high applied voltage, thus producing current decrease or biphasic pulse signals. These results were further confirmed by the gel electrophoresis (Fig. 3B). The control experimental result demonstrates that only one band is observed at very low position in the absence of target DNA, confirming that the two hairpin probes stably coexist in the solution (lane 7). However, a series of new bright stripes are generated in the presence of various ratios of [I0]/[H1], indicating that the HCR reaction have taken place and the two auxiliary probes had been assembled to long-nicked DNA concatemers (lane 1-6). Interestingly, the position of these stripes, corresponding to the molecular weight of assembly products, changes with the concentration of H1 and H2 when the concentration of target DNA was constant. At the high ratios of 1:10 and 1:20, the molecular weight of longnicked DNA polymers is relatively small since plentiful target DNA compete the hairpin DNAs and restricted the assembly degree of HCR (lane1-2). When the ratio decreases to 1:100 or lower (lane 3-4), the stripes at lower position became dim, while new bright stripes are observed at higher position. The findings suggest that the DNA polymerization reaches the maximum degree and the molecular weight of DNA concatemers were relatively large. However, the molecular weight of self- assembled DNA concatemers are slightly decreased when the concentration of H1 and H2 increases from 10 µM to 20 µM (lane 5-6), it could be explained by a reported “inhibitor ultrasensitivity” mechanism (Li et al. 2012). It is worth to mention that the molecular weight of the HCR products were an approximate range, not an exact numerical value (Liu et al. 2013). Therefore, multiple pulse signals could be produced during the translocation of the products. The dependence of the event frequency on the ratio of [I0]/[H1] is shown in Fig. 3C. Interestingly, the characteristic signals frequency exhibits a non-monotonic dependence with decreasing the ratio of [I0]/[H1]. As the [I0]/[H1] decreases from 1:10 to1:100, the event
frequency increases from 48 ± 2.3 to 58 ± 2.7 min-1. Then a gradual decrease is observed in the range of 1:100 to 1:2000. In addition, the effect of the ratio of [I0]/[H1] on current pulse amplitude was also examined. As shown in Fig. 3D, the pulse amplitude first shows a slight increase and then remains almost constant as an increase in the ratio. These results could be explained by the fact that the molecular mass of the DNA concatemers products reaches a summit at a specific ratio of [I0]/[H1], 1:100. This interpretation can be also supported by the gel electrophoresis experiment as shown in Fig. 3B. In order to achieve the detection of target DNA with high sensitivity and resolution, the ratio of 1:100 is chosen as the optimized condition and used in the remaining experiments.
Figure 3 should be inserted in here
3.4. Detection sensitivity and specificity Fig. 4A is representative current traces at various target DNA concentrations. The frequency of characteristic signals increases with an increase in the concentration of target DNA in a wide range of 20 fM to 5 pM (Fig. 4B). Linear regression analysis exhibits good linearity in the range of 50 fM-1.0 pM, with a correlation coefficient of 0.994 (Fig. 4B, inset). The limit of detection is 5.2 fM (S/N = 3). To evaluate the selectivity, a series of comparative studies using single-base mismatch (MT1), two-base mismatch (MT2) and non-complementary (NC) sequences were investigated as control experiments. As illustrated in Fig. 4C, only fully matched DNA (TD) initiates the HCR and nanopore responses, while the other mismatched DNAs are difficult to trigger the reaction and to generate significant current pulse signals. The high selectivity with
single-base resolution should originate from HCR that requires full complementarity between the target DNA and hairpin auxiliary probes (Liu et al. 2013). In addition, the sequence specificity of this DNA assay was further validated by gel electrophoresis (Fig. 4D).
Figure 4 should be inserted in here
3.5. ctDNA detection It is well known that the release of KRAS G12DM, a single-stranded ctDNA into the blood is related to the apoptosis and necrosis of cancer cells in tumor tissues (Fig. 5A), and its level in blood can be used to evaluate the progression of colorectal cancer (Schwarzenbach et al. 2011). Fig. 5B shows the process of detecting KRAS G12DM ctDNA. According to KRAS G12DM sequence (see Table S1), two new hairpin auxiliary probes H3 and H4 (the sequences see Table S1) were designed, which can form long dsDNA polymers through HCR in the presence of KRAS G12DM target DNA. As observed in Fig. 5C, the characteristic signals are generated in the range of 20 fM to 2 pM. Moreover, the full matched KRAS G12DM target can be well discriminated from that of one-base, two-base and three-base mismatched DNA sequences (see Table S1 and Fig. S2 in Supporting Information). The limit of detection (LOD) was 2.8 fM, with a linear range of 20 fM-1 pM (Fig. 5D, inset). The linear regression equation can be expressed as freq=82.31[ctDNA] (pM) + 1.84 (R = 0.995). In addition, despite multicomponent (proteins and ctDNA) in serum sample, no signal was observed in the commercial serum sample (Figure. S3). This result could be attributed to a fast translocation of each individual component and thus the event signals can hardly be captured.
Figure 5 should be inserted in here
3.6. Serum Sample Assay We explored the application for detecting ctDNA in real serum. 10-fold the diluted commercial healthy human serum and the serums with added target KRAS G12DM ctDNA (100 fM, 500 fM and 1 pM) were tested by our method (Fig. S3). The detected concentrations are 90.9 ± 3.12 fM, 475.0 ± 16.76 fM and 931.1 ± 38.83 fM, respectively. The corresponding recoveries are 90.92 ± 3.12%, 95.0 ± 3.35% and 93.1 ± 3.88%, exhibiting adequate and acceptable accuracy and anti-interference ability for ctDNA detection (Table S2). 4. Conclusions Overall, we have successfully constructed HCR-nanopore sensing system and applied in the sensitive detection of ctDNA. The HCR can be harnessed in solid nanopore sensing, overcoming the limitation that has precluded the short DNA or small target. The HCR amplification is a key for obtaining high sensitivity and high selectivity. The amplitude and frequency of nanopore signal can be modulated in accordance with the target by the probe concentration. This simplest single-molecule analytical technique is very suitable for monitoring ctDNA level in circulating system that needs readout the result quickly because of short the half-life. Our approach could be further improved through single-stranded DNA libraries (Burnham et al. 2016). Combined with PCR-free libraries, the HCR-nanopore could reveal the molecular features and the origin characteristics of ctDNA that are distinct at different positions such as nucleosome positions, the start sites and the end positions of transcription (Jiang et al. 2018). Additionally, the solution-
based technology required only a small amount of blood sample (<10 µL), which should be broadly employed in the screening and management in clinical routine test.
CRediT authorship contribution statement Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was financially supported by the National Science Foundation of China (Grants 21375104, 21327806 and 21874107), and Natural Science Basic Research Plan in Shaanxi Province of China (Grant 2016JQ2021).
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Fig. 1. (A) Schematic illustration of the HCR-nanopore biosensor for DNA detection. Through the hairpin probes of H1 and H2, HCR amplifies short target DNA to form long-nicked dsDNA concatemer, followed by nanopore sensing. H1, H2 and the target were added to the base side of nanochannel. (B) Event frequency values of target DNA (black), H1/H2 (red) and DNA concatemers (blue) at different voltages. (C) SEM image of the base side of conical PET nanochannel. (D) I-V characteristics of the conical nanochannel measured in 10 mM Tris-HCl buffer (pH 7.0) containing 1 M KCl.
Fig. 2. (A) The schematic of DNA concatemers translocation processes (top), the expanded view of three typical translocation event signals (bottom). (B) The histogram of the current changes. (C) The histograms of the dwell time for four levels (Ⅰ, Ⅱ, Ⅲ and Ⅳ, as denoted in Fig. 2A). Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied voltage of - 1.0 V.
Fig. 3. (A) Typical current-time traces at various ratios of [I0]/[H1] (1:10 to 1:2000). (B) The image of gel electrophoresis (M: marker; lanes 1-6: the concentration of target DNA is 10 nM, the concentrations of H1 and H2 all are 100 nM, 200 nM, 1 µM, 5 µM, 10 µM, 20 µM, respectively; lane 7: 20 µM H1 + 20 µM H2). (C-D) The effect of molar concentration ratios of [I0]/[H1] on the frequency (C) and amplitude (D). Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied potential of - 1.0 V.
Fig. 4. (A) Representative current traces of target DNA at various concentrations. (B) The plots of translocation event frequency versus the target DNA concentrations. Inset is the linear relationship between the event frequency and the target DNA concentration in the range of 50 fM to 1 pM. (C) Selectivity of the method for target DNA detection. The concentration of target DNA and the mismatched DNA sequences (see Table S1) are 1 pM and 10 pM, respectively. (D) Agarose electrophoresis images (M: marker; lane 1: H1 + H2; lane 2: H1 + H2 + target DNA; lane 3: H1 + H2 + MT1; lane 4: H1 + H2 + MT2; Lane 5: H1 + H2 + NC. The concentration of target sequence and auxiliary probes were 10 nM and 1 µM, respectively).
Fig. 5. (A) Schematic illustration of circulating tumor DNA fragments releasing from cells and circulating in the blood. (B) The analysis process of KRAS G12DM ctDNA. (C) The typical current traces of KRAS G12DM ctDNA with different concentrations. (D) Dose-response curve of KRAS G12DM ctDNA. Inset is the linear calibration curve. Experimental conditions: 1.0 M KCl and 10 mM Tris-HCl (pH 7.0), with the applied potential of - 1.0 V.
Highlights
1. We have constructed HCR-nanopore sensing system and applied in the detection of ctDNA. 2. The sensing strategy exhibited well selective and the detection limit is 2.8 fM. 3. The proposed method can apply in detection of ctDNA in serum sample. 4. The method potentially offers a simple and non-invasive liquid biopsy for diagnosing cancer.
Author Contribution Statement
Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
CRediT authorship contribution statement Hong Sun: Methodology, Writing - original draft. Fujun Yao: Writing - review & editing. Zhuoqun Su: Discussion and analysis. Xiao-Feng Kang: Writing -review & editing, Supervision.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: